Beta-lactam antibiotics are among the most important clinically, and the isolation of novel .beta.-lactam compounds continues six decades after the discovery of the penicillins by Fleming. The common structural feature of the penicillins and cephalosporins (including cephamycins) is the .beta.-lactam ring.
These antibiotics are produced by a variety of prokaryotes and lower eukaryotes. The penicillins, exemplified by the compounds penicillin G (benzyl-penicillin) or penicillin V, are produced by filamentous fungi, most notably Penicillium chrysogenum. The cephalosporins, first isolated as a product from the lower eukaryote, Cephalosporium acremonium (syn. Acremonium chrysogenum), are also metabolites of many prokaryotes, especially Streptomyces clavuligerus, S. lipmanii and S. cattleya, that also produce cephamycins and other .beta.-lactams such as oxypenams (clavulanic acid) and carbapenems (thienamycin).
The development of cell-free systems from .beta.-lactam-producing organisms has allowed the establishment of the biosynthetic steps in the pathway of the sulfur-containing .beta.-lactams (penicillins and cephalosporins).
The initial steps in the formation of penicillins in filamentous fungi (e.g., P. chrysogenum), and the cephalosporins produced by both prokaryotes (e.g., S. clavuligerus) and lower eukaryotes (e.g., C. acremonium), are identical. ACV synthetase catalyzes the condensation of the amino acid precursors L-.alpha.-aminoadipate, L-cysteine, and L-valine to the tripeptide LLD-ACV. The next step forms the first .beta.-lactam in the pathway by the cyclization of the tripeptide yielding isopenicillin N (IPN), a precursor to all penicillins, cephalosporins and cephamycins.
After synthesis of IPN, the pathways to cephalosporins and penicillins diverge. In P. chrysogenum, for example, the .alpha.-aminoadipyl side chain of IPN can be exchanged for one of many (nearly 100 to date) hydrophobic side chains derived from the corresponding acyl CoA. One of the most familiar examples is the formation of penicillin G (benzylpenicillin) from phenylacetyl CoA and IPN. However, in the fungus C. acremonium, the .alpha.-aminoadipyl side chain is isomerized to produce penicillin N. The five-membered thiazolidine ring of the penicillin is then "expanded" to the six-membered dihydrothiazine ring that is characteristic of the cephalosporins. This reaction is catalyzed by deacetoxycephalosporin C synthetase (DAOCS) and produces the first cephalosporin in the pathway, deacetoxycephalosporin C (DAOC).
The present invention provides DNA compounds comprising an isolated DNA sequence encoding an activity involved in the production of penicillins and cephalosporins, said activity being referred to as .delta.-(L-.alpha.-aminoadipyl)-L-cysteinyl-D-valine (ACV) synthetase. The present invention expands the repertoire of beta-lactam biosynthetic enzymes which can be overproduced. This ability facilitates the bioconversion of substrate analogs to novel beta-lactams and strain improvement by increased gene dosage.